Increased amyloid [unreadable] (A[unreadable]) 42/40 ratio has been reported in aging brain and in Alzheimer's disease (AD), and is linked to memory loss and AD pathogenesis. The final enzymatic step in generating Ass via cleavage of the amyloid precursor protein (APP) is performed by the presenilin 1 (PS1) dependent ?-secretase complex. The precision of the PS1/?-secretase cleavage, thus, determines which A[unreadable] species will be produced: Ab40 or highly fibrillogenic and neurotoxic A[unreadable] 42. Importantly, over 150 known mutations in PS1, scattered throughout the molecule, all lead to autosomal dominant familial AD (FAD) and enhance A[unreadable] 42 generation. Thus, regulation of the A[unreadable] 42/40 ratio are of particular therapeutic importance. Recent data from several laboratories, as well as our own, support the possibility that subtle changes in PS1 conformation shift the ?-secretase cleavage site on APP, and thus affect the A[unreadable] 42/40 ratio. Since PS1 conformation correlates with the A[unreadable] 42/40 ratio, finding compounds that allosterically modulate PS1/g-secretase, as oppose to inhibit ?-secretase function, would provide an attractive therapeutic strategy. Moreover, February, 2010 meeting in Phoenix, AZ, discussed the Alzheimer's Prevention Initiative (API). The idea is to start testing candidate drugs in people who are at imminent risk for Alzheimer disease but have no symptoms. These are FAD PS1 E280A mutation carriers, members of a large Colombian family. The concern is, however, that some FAD PS1 mutations could be resistant to a particular drug (Czirr et al., 2008). Thus, having an assay that will allow to screen for allosteric modulators of PS1/?-secretase without inhibiting its function, and to test the potency/dose dependence/toxicity etc. of a lead compound would be crucial for development of FAD-based therapeutics. Our observations, using a novel FRET based techniques in intact cells support a model with the following important, testable features: FAD PS1 mutations bring PS1 N- terminus (NT) and loop domain close together, and this represents a pathogenic change in conformation associated with increased A242 production due to altered alignment of the PS1/?-secretase active site with APP substrate. We have recently developed a novel molecular reporter probe, GFP-PS1-RFP (G-PS1-R), which allows monitoring intra-molecular interactions in intact and/or live cells. Our preliminary data using cell-by-cell microscopy approaches show that both genetic and pharmacological manipulations that affect the A[unreadable] 42/40 ratio consistently change G-PS1-R conformation (NT-loop proximity). We propose to configure this FRET-based assay to a high-throughput format to 1) test numerous FAD PS1 mutations for their ability to alter PS1 conformation, and 2) to use this assay to screen Molecular Libraries for allosteric modulators able to "correct" abnormal pathogenic PS1 conformation associated with elevated A[unreadable] 42/40 ratio. If successful, this project will help to identify candidate therapeutic leads and evaluate their efficacy, thus providing a proof-of-principle concept for novel therapeutic strategy to prevent or cure AD, which may even slow down some deleterious effects of aging. PUBLIC HEALTH RELEVANCE: Due to the increased average population age, and thus increased number of affected individuals and associated tremendous healthcare costs, AD has become an urgent public health concern in the U.S. The unmet medical need for developing novel and effective AD therapeutics has been recognized by NIH. We propose to develop and validate an HTS assay to monitor PS1 conformation in intact/live cells. This assay will be used to identify compounds, allosteric modulators of the PS1/?-secretase able to "correct" abnormal pathogenic conformation associated with increased generation of the neurotoxic A242 species. If successful, this project will help to identify candidate therapeutic leads and evaluate their efficacy, thus providing a proof-of-principle concept for novel therapeutic strategy to prevent or cure AD, which may even slow down some deleterious effects of aging.